Chromium single cell 3 reagent kit

Single-cell suspensions were counted using the Vi-Cell XR Cell Viability Analyzer (Beckman-Coulter, Brea, CA, USA) and ~5000 cells were loaded into the 10X Genomics Chromium system to generate Gel Beads-In-Emulsion (GEMs). Reverse transcription was performed using Chromium Single Cell 3’ Reagent Kits (v2, 10X Genomics, Pleasanton, CA, USA). Standard Illumina sequencing primers and a unique i7 Sample index were added to each cDNA for library construction. Libraries were sequenced using the Illumina cBot and HiSeq 3000/4000 PE Cluster Kit. The flow cells were sequenced as 100 × 2 paired-end reads on an Illumina HiSeq 4000 using HiSeq 3000/4000 sequencing kit and HCS v3.3.52 collection software. Base-calling was performed using Illumina’s RTA version 2.7.3. Cell Ranger (v3.0) (10X Genomics) was used for alignment to the mouse genome version mm10. R (v3.6.0) and Seurat (v3.1.3)^{35 (link)} were used for further analysis following the standard workflow. For quality control and filtering, cells with detected genes fewer than 200 or >8000, or with mitochondrial gene content >50% were excluded. For Ximerakis’s scRNA-seq dataset, R (v4.0.3) and Seurat (v4.0.2) were used. Gene set enrichment analysis was performed using software GSEA (v4.0.3)^{53 (link)}. The interactive website for scRNA-seq data was generated using ShinyCell (v2.1.0)⁵⁴ .

Libraries were generated with 10x Genomics (USA) Chromium single-cell 3′ reagent kits according to the manufacturer’s instructions. Version 2 reagent kits were used for donors R253 and R282, and Version 3 was used for donor R317. Each experimental condition was labelled as a sample, with a total of three samples per donor. Completed libraries were pooled and sequenced on the Illumina (USA) NextSeq500.

For the construction of snRNA-seq libraries, Chromium Single Cell 3′ Reagent Kits (10X Genomics; v.2 chemistry for human, chimpanzee, bonobo, gorilla, gibbon, macaque, marmoset, mouse and opossum; v.3 chemistry for platypus and chicken) were used according to the manufacturer’s instructions. Then 15,000 to 20,000 nuclei were loaded per lane in the Chromium microfluidic chips and complementary DNA was amplified in 12 PCR cycles. Sequencing was performed with NextSeq550 (Illumina) according to the manufacturer’s instructions using the NextSeq 500/550 High Output Kit v.2.5 (75 cycles) with paired-end sequencing (read lengths of read1 26 bp, read2 57 bp; index1 8 bp, roughly 170 to 380 million reads per library for v.2 chemistry; read lengths of read1 28 bp, read2 56 bp Index1 8 bp, roughly 247 to 306 million reads per library for v.3 chemistry) (Supplementary Table 2).
For bulk RNA-seq data generation, RNA was extracted using the RNeasy Micro kit (QIAGEN). The tissues were homogenized in RLT buffer supplemented with 40 mM DTT. The RNA-seq libraries were constructed using the TruSeq Stranded messenger RNA LT Sample Prep Kit (Illumina) as described in ref. ^{2 (link)}. Libraries were sequenced on Illumina NextSeq550 using a single-end run (read1 159 bp; index1 7 bp) with roughly 24–60 million reads per library (Supplementary Table 2).